The investigation seeks to establish definitive evidence for the function of a guanine nucleotide regulatory protein in a pathway of cellular catecholamine action different from that associated with activation or inhibition of adenylate cyclase. The mechanisms of alpha1-adrenergic action will be studied in the cultured rat myocardial cell. This cell converts its response to alpha1 agonist stimulation from an increase to a decrease in rate of beating in association with the functional acquisition of a 41K ADP-ribosylatable substrate for pertussis toxin. The specific aims are to define the role of the pertussis toxin substrate in the chronotropic effects of the alpha1-adrenergic catecholamines in cultured cardiac cells; and to establish the role of the pertussis toxin substrate in the actions of the alpha1-adrenergic catecholamines to stimulate phosphatidylinositol turnover and to increase cytosolic calcium concentrations in myocytes. The effect of alpha1 agonist to stimulate phosphatidylinositol turnover and to activate the enzyme, phospholipase C, implicated as the site of alpha1 agonist action, will be determined. Cytosolic calcium will be measured by fluorescent probes in suspended cells and by fluorescence microscopy of isolated single myocytes. The second major objective is to purify a cytosol regulator of adenylate cyclase activity obtained from rat reticulocytes, and to determine its mechanism of action. Reticulocyte cytosol activator protein (RCAP) augments adenylate cyclase activity at the level of the guanine nucleotide regulatory proteins. RCAP will be purified to homogeneity. The site at which and mechanisms by which RCAP interacts with the guanine nucleotide proteins will be established. The major effect of RCAP upon the pertussis toxin substrate, Ni, observed in all tissues studied so far, suggests a potential role for RCAP in the regulation of the non-cyclic AMP associated pathway of alpha1-adrenergic action to be studied in the myocardial cell. Methods to be employed include new biochemical approaches to protein purification, isolation of the guanine nucleotide binding proteins, GTP binding and GTPase assays of the alpha subunits of N under the influence of RCAP. Further understanding of the cellular actions of the catecholamines resulting from these investigations is likely to provide a framework with which abnormalities of catecholamine function can be more completely appreciated.